Systemic exposure to ionizing radiation (IR) results in cell damage in radiosensitive tissues. It is therefore highly desirable to develop efficient radioprotective agents for use in cancer radiotherapy, and biodefense. We have discovered a new class of radioprotective compounds derived from bacterial polypeptides that act as agonists of Toll-like receptors (TLRs). Our lead compound, CBLB502, binds to TLR5 and activates the NF-kB pro-survival pathway, which protects from IR damage. Phase I of this proposed research aims to improve CBLB502 and to identify novel radioprotective compounds from peptides that induce the NF-kB pathway by screening the libraries of BioActive Secreted Proteins (BASP). We will also derive high affinity CBLB502 BASP variants by mutagenesis. Potential radioprotective peptides that do not act through NF-kB will be isolated by direct functional selection for radiation protection. In addition, completion of Phase I of the proposed research will result in the development and commercialization of a new BASP screening technology, based on the in silico design and construction of short peptide BASP libraries with full control of amino acid residues, which is compatible with functional screening in disease-relevant cell models. This method was made possible by recent innovations in high-throughput (HT) chip-based oligonucleotide synthesis and HT sequencing technology. This simple and cost-effective approach represents an improvement on current drug discovery strategies and will accelerate the development of novel peptide therapeutics. Under Phase I funding, we propose to develop and validate this novel resource in vitro and in vivo. Given our unique interface with the cell-based drug screening facilities at the Roswell Park Cancer Center, we expect to develop a pipeline of potential drug peptide mimics with fast conversion of knowledge into efficient strategies of drug identification. Phase II of this research proposes an in-depth characterization of the isolated peptides as potential drugs. At the completion of the proposed studies, we anticipate to generate a novel class of radioprotective drugs. We believe the development of BASP screening platform will provide a valuable tool set for the identification and validation of novel biologically relevant peptide drugs. It will help to identify novel targets for therapeutic intervention and establish paradigms for drug discovery. Most importantly, this project will expedite the transition from bench to clinic and provide both academic and industrial researchers with a robust, cost-effective alternative to current peptide screening strategies. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify novel radioprotective agents for biodefense and cancer treatment by peptide screening. The libraries of bioactive secreted peptides will be functionally screened for suppression of radiation-induced cell death and validated in vivo.